Myosins are the only known actin-based motors and many genes encoding myosins have been identified. Few myosins have been studied in enough depth to understand their functions in cells. Some myosins move along actin to transport cellular components or create tension. Myosin VI is an unusual myosin because in moves in a direction opposite to most other myosins - it moves toward the pointed or slow growing end of an actin filament. It also binds tightly to actin and takes very large steps. The characteristics suggest it may have a very different function than other family members. Myosin VI mutants in vertebrates cause deafness due to degeneration of the sensory epithelia. The primary defect in this tissue is in the actin-based stereocilia if the hair cells. These structures are important for sensing sound. Mutations in myosin VI in Drosophila are lethal, suggesting it plays a unique and important role in vivo. Myosin VI has been shown to bind to several membrane-associated proteins and be involved in a number of processes that require actin-membrane interaction in vertebrates and model organisms such as Drosophila and C. elegans. It is not clear how loss of myosin VI causes the observed defects. However, understanding the mechanism by which myosin VI works in cells is important to understanding and eventually repairing defects caused by myosin VI loss of function. We are using Drosophila as a model system to investigate myosin VI's role in vivo. We are primarily examining myosin function during spermatogenesis, where it is important for an actin-mediated membrane remodeling process called spermatid individualization. We have evidence that myosin VI regulates actin assembly. Actin assembly is important for movement of the actin structure that mediated membrane remodeling, the actin cone. This motility process has some similarities to and differences from other wellstudied motility processes, like motile cell leading edge protrusion. Actin assembly regulation is a previously unexpected role for myosin VI. In this proposal we plan to investigate the mechanism by which myosin VI regulates actin assembly and how this regulation leads to normal actin cone movement. We plan to use biochemical, immunocytochemical, molecular genetic and classical genetic methods to determine which proteins work with myosin VI during individualization. We have proposed a model for how the actin structures involved in individualization move and will test various predictions of this model. In addition, we will examine myosin VI function in another process - secretion of salivary gland glue granules - to determine if its mechanism of action is similar. We also have preliminary evidence that myosin VI associates with tissue-specific light chain related to calmodulin. Light chains of other myosins regulate activity. Some data from assays of myosin VI's enzymatic properties in vitro support the idea that it, too, is regulated through its light chain. We propose to investigate light chain interactions. In the long term, we hope to examine associations with light chains, phosphorylation, motility and other properties of myosin VI to understand their importance in vivo. Myosin VI is likely to be important in many cell types for actin-based processes. Since myosin VI and other elements of the actin cytoskeleton are conserved across multicellular animals, what we learn should be widely applicable.